In anatomical pathology, thin tissue sections are mounted on microscope slides, and stained with a combination of stains. Each stain has a characteristic hue (e.g., Hemotoxylin is blue-purple., Eosin is red-pink, DAB is brown), and is used to visualize a particular cellular component or bio-marker. A plurality of stains, each with its own color, is often used on the same slide. The slide is typically imaged with a camera or scanner that provides red, blue, and green intensities for each pixel in the image. Each pixel represents a corresponding location on the slide.
One goal of automated processing of such slides measure the intensity of each dye at each location in the slide by using the “known” color pattern of each dye in terms of intensities of red, green, and blue light emitted by that dye. The resultant pixel values define a vector in a three-dimensional space whose axes are the intensities in red, green, and blue color bands. To simplify the following discussion, two pixels whose color vectors can be made equal to one another by multiplying each of the components of one of the color vectors by a constant will be defined to have the same hue. Each stain gives rise to pixels having similar hues. Unfortunately, the stain hues are known to vary between slides for the commonly used dyes.
To correct for this variation, a calibration step in which similar tissue sections are singly stained with each stain, and visualized to determine the pure spectra of each stain are used. However, in addition to the additional labor, this type of calibration procedure presents challenges if the individual stains interact with one another so as to produce a spectrum shift in the region of overlap.